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Galectin-3 (Gal-3) is a biomarker involved in fibrosis and vascular inflammation. Gal-3 has been linked to chronic kidney disease (CKD) and patients with peripheral artery disease (PAD). Conflicting reports exist about the relevance of Gal-3 in PAD. The study aims to elucidate a possible link between serum and urinary Gal-3 and long-term survival in PAD patients without critical limb ischemia and mild to moderate CKD.Galectin-3 (Gal-3) was measured in serum (n = 311) and urine (n = 266) of PAD patients (age 69 (62-77) years) by bead-based multiplex assay. Urinary Gal-3 concentration was normalized to urine creatinine (cr) levels. Mortality data were retrieved from the Austrian central death registry after a median observation period of 9.2 years. Survival analyses were performed by the Kaplan-Meier method and Cox-regression.Serum Gal-3 was higher in patients with claudication symptoms (p = 0.001) and correlated inversely with the patients' ankle-brachial index (R = -0.168, p = 0.009). Serum Gal-3 and urinary Gal-3 (uGal-3/cr) were associated with the estimated glomerular filtration rate (R = -0.359, p 0.001; R = -0.285, p 0.001). Serum Gal-3 was not linked to all-cause mortality [HR 1.17 (CI 0.96-1.42)] over 9.2 years. However, uGal-3/cr was associated with all-cause mortality [HR 1.60 (CI 1.31-1.95)]. This association sustained multivariable adjustment for cardiovascular risk factors and renal function [HR 1.71 (CI 1.35-2.17)].This study is the first to show an association of uGal-3/cr and long-term mortality in patients with PAD. Gal-3 was not predictive of long-term mortality but seems to be a marker of PAD severity in patients without critical limb ischemia.

We evaluated angiogenin as a prospective biomarker in peripheral artery disease (PAD) patients with and without claudication symptoms. A pilot study suggested an elevation of angiogenin in critical limb ischemia. However, in PAD patients, the predictive value of angiogenin has not yet been evaluated. For this purpose, 342 patients with PAD (age: 69 ± 10 years, 34.5% women) were followed-up for 7 years in a cross-sectional study. Angiogenin was measured by enzyme-linked immunosorbent assay. All-cause and cardiovascular mortality were analyzed by Cox regression. Angiogenin levels were higher in men ( P = .001) and were associated with patient waist-to-hip ratio ( P < .001), fasting triglycerides ( P = .011), and inversely with estimated glomerular filtration rate ( P = .009). However, angiogenin showed no association with age, characteristics of diabetes, markers of lipid metabolism, or C-reactive protein. Angiogenin did not correlate with markers of angiogenesis such as vascular endothelial growth factor, angiopoietin-2, or tie-2. Furthermore, angiogenin was not associated with PAD Fontaine stages or with patient ankle-brachial index in addition to all-cause mortality (hazard ratio [HR] = 1.09 [95% CI: 0.89-1.34]) or cardiovascular morality (HR = 1.05 [0.82-1.35]). These results suggest that angiogenin does not provide further information regarding outcome prediction in patients with PAD.

Background and aims The TNF-superfamily member sTWEAK and its scavenger receptor sCD163 are potentially involved in pathophysiological processes of atherosclerosis. In patients with peripheral arterial disease, previous research has shown that sTWEAK and the sCD163/sTWEAK ratio were independently associated with long term all-cause and cardiovascular survival. Since previous investigations emphasized on symptomatic peripheral arterial disease including critical limb ischemia, this study evaluates sTWEAK and sCD163 in a cohort of stable peripheral arterial disease including asymptomatic (Fontaine stage I) and intermittent claudication (Fontaine stage II) patients. Methods sTWEAK concentrations of 354 patients were measured using a commercially available ELISA kit. sCD163 was quantified using a multiplex bead assay. Cox proportional hazards regression was used to assess outcome after a seven-year follow-up. Hazard ratios are given as interquartile range. Results Patients with intermittent claudication exhibited increased sCD163 levels in comparison to asymptomatic patients (p = 0.002). However, sTWEAK was not related to peripheral arterial disease severity (p = 0.740). A multivariable Cox-proportional hazard models including sTWEAK and cardiovascular risk factors (age, HbA1c, CRP, LDL-C, BMI, eGFR) revealed an inverse association with all-cause mortality (HR 0.775 (95% CI 0.623–0.965) and cardiovascular mortality (HR 0.710 (95% CI 0.534–0.944)). Further multivariable models including sCD163 or the sCD163/sTWEAK ratio and cardiovascular risk factors showed no association with mortality. Conclusions This study highlights the use of sCD163 as a novel biomarker for PAD severity and supports sTWEAK as an independent predictor of all-cause and cardiovascular mortality even in stable peripheral arterial disease.

Thrombospondin-4 (TSP-4) is an extracellular matrix protein of the vessel wall. Despite bench evidence, its significance in the clinical setting of atherosclerosis is missing. TSP-4 (ng/ml) was measured in 365 PAD patientsusing a commercially available ELISA. PAD was diagnosed by the ankle-brachial index (ABI) and clinically graded using the Fontaine classification. TSP-4 levels were significantly higher in Fontaine II vs. Fontaine I (4.78 ± 0. 42, 4.69 ± 0.42, p = 0.043). TSP-4 significantly correlated with ABI (r = - 0.141, p = 0.023, n = 259) after the exclusion of mediasclerotic patients. Binary logistic regression analysis for Fontaine I vs. II showed an OR of 1.70 (1.02-2.82) in a multivariable model adjusted for traditional risk factors. Interestingly, TSP-4 levels were higher in patients with type 2 diabetes mellitus or prediabetes (DGT) compared with normal glucose tolerance (NGT) (4.76 ± 0.42 vs. 4.66 ± 0.41, p = 0.035). ANOVA for PAD and diabetes subgroups showed a linear increase with disease burden with the highest difference between Fontaine I-NGT and Fontaine II-DGT (4.59 ± 0.40, 4.79 ± 0.43, p = 0.015). TSP-4 levels increased with PAD severity and showed a former unknown association with diabetes. Thus, TSP-4 could be a novel marker of atherosclerotic activity, especially in the major subgroup of patients with concomitant diabetes.

YKL-40 is an inflammatory marker secreted by macrophages and is expressed in atherosclerotic plaques. YKL-40 increases in coronary artery disease (CAD) with poor coronary collateral vessel development. Higher levels are linked to reduced survival in CAD patients. Studies evaluating YKL-40 in patients with peripheral arterial disease (PAD) are scarce. This study aims to elucidate a possible link between YKL-40 and PAD severity as well as cardiovascular long-term mortality.YKL-40 was measured at baseline in 365 elderly PAD patients (age 69 ± 10.4, 33.7% women, Fontaine stage I-II) by bead-based multiplex assay. Patients were followed for seven years to assess long-term cardiovascular and all-cause survival by Kaplan-Meier and Cox regression.YKL-40 levels were associated with declining ankle-brachial index (ABI) in PAD patients without Moenckeberg's mediasclerosis (R = -0.189, p=0.002). PAD patients with mediasclerosis exhibited higher YKL-40 levels (p=0.002). Baseline YKL-40 levels were significantly associated with cardiovascular mortality (HR 1.52 (1.21-1.91), p 0.001) and all-cause mortality (HR 1.45 (1.20-1.75), p 0.001) over a seven-year observation period. After multivariable adjustment for gender, patient age, known carotid artery disease, known coronary artery disease, smoking status, systolic blood pressure, HbAIncreased YKL-40 levels are independently associated with poor long-term cardiovascular survival in peripheral arterial disease patients. Furthermore, YKL-40 correlates with patients' ABI in PAD in the absence of mediasclerosis.

Survival of peripheral arterial disease (PAD) patients increased over the last decade due to increased use of secondary preventive medication and rapid revascularization of PAD patients. Angiogenetic markers such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2) and its receptor Tie-2 might be useful markers to assess the residual risk for mortality in PAD patients. The aim of this study was to evaluate angiogenetic markers for the prediction of mortality in a PAD cohort. For this purpose, 366 patients (mean age: 69 ± 10 years) with PAD Fontaine stage I or II were included and followed up over a 5-year study period. Serum Ang-2, Tie-2 and VEGF levels were measured by bead-based multiplex assay. All-cause mortality and major cardiovascular events (MACE) including all-cause death, non-fatal stroke and non-fatal myocardial infarction were analysed by Kaplan–Meier and Cox regression analyses after 5 years. Ang-2 was associated with Tie-2 (R = 0.151, p = 0.006) and VEGF levels (R = 0.160, p = 0.002). However, only Ang-2 was linked to all all-cause mortality in PAD patients (hazard ratio [HR]: 1.55 [1.23–2.15], p = 0.008) even after adjustment for age and gender, haemoglobin A1c, low-density lipoprotein cholesterol, systolic blood pressure and glomerular filtration rate (HR: 1.44 [1.03–2.00], p = 0.032). Furthermore, an association of Ang-2 and MACE in PAD patients (HR: 1.36 (1.03–1.78), p = 0.028) was found. This result implies that Ang-2 might be used as an additional marker to stratify PAD patients to predict poor mid-term life expectancy.

Fatty acid–binding protein 4 (FABP4) is a possible biomarker of atherosclerosis. We evaluated FABP4 levels, for the first time, in patients with peripheral artery disease (PAD) and the possible association between baseline FABP4 levels and cardiovascular events over time. Patients (n = 327; mean age 69 ± 10 years) with stable PAD were enrolled in this study. Serum FABP4 was measured by bead-based multiplex assay. Cardiovascular events were analyzed by FABP4 tertiles using Kaplan-Meier and Cox regression analyses after 5 years. Serum FABP4 levels showed a significant association with the classical 3-point major adverse cardiovascular event (MACE) end point (including death, nonlethal myocardial infarction, or nonfatal stroke) in patients with PAD ( P = .038). A standard deviation increase of FABP4 resulted in a hazard ratio (HR) of 1.33 (95% confidence interval [95% CI]: 1.03-1.71) for MACE. This association increased (HR: 1.47, 95% CI: 1.03-1.71) after multivariable adjustment ( P = .020). Additionally, in multivariable linear regression analysis, FABP4 was linked to estimated glomerular filtration rate ( P < .001), gender ( P = .005), fasting triglycerides ( P = .048), and body mass index ( P < .001). Circulating FABP4 may be a useful additional biomarker to evaluate patients with stable PAD at risk of major cardiovascular complications.

Background and aims Previous data show contradicting results regarding relevance of obesity on outcome in peripheral arterial disease (PAD). Thus, this study aims to evaluate the predictive power of obesity as measured by established and novel obesity indices (waist circumference WC, waist-hip ratio WHR, body-mass index BMI, body adiposity index BAI, visceral adiposity index VAI, weight-adjusted waist index WWI) in a PAD cohort. Methods and results In 367 patients with diagnosed PAD anthropometric parameters were assessed at study inclusion in an observational study. Mortality data was retrieved from the central death registry after five years. Outcome analyses were performed by multivariable Cox-regression models. 57 PAD patients (15.5%) died during the follow-up, of those 36 were categorized as cardiovascular origin. Patients from the all-cause mortality group were older, more often diabetics with a worse glucose control and had worse renal function. Obesity indices were not significantly different between the event and control group. None of the evaluated risk factors predicted cardiovascular or all-cause death after multivariable adjustment for age, gender, LDL-C, serum creatinine, systolic blood pressure, CRP, smoking habits, diabetes status and previous history of peripheral revascularisation (all-cause WC 1.007 (0.983–1.031), WHR 1.772 (0.106–29.595), BMI 1.006 (0.939–1.078), BAI 1.002 (0.945–1.063), VAI 1.019 (0.895–1.161), WWI 1.085 (0.831–1.416); cv-death WC 1.007 (0.978–1.036), WHR 0.382 (0.006–25.338), BMI 1.004 (0.918–1.098), BAI 1.034 (0.959–1.116), VAI 1.036 (0.885–1.213), WWI 1.061 (0.782–1.441)). Conclusion Obesity as risk marker estimated by indices both for general and visceral adiposity, does not predict mortality in a secondary prevention cohort of PAD patients.

Background and introduction Endothelial dysfunction and associated cells are an important cornerstone in the development and progression of peripheral artery disease (PAD). Endothelial progenitor cells (EPC) are released from the bone marrow and have exhibited the potential for cardiovascular repair. Higher EPC levels have been linked to longer event-free survival in coronary artery disease. Similar evaluation of EPC on mortality in PAD is lacking. Purpose The current study aimed to evaluate the possible association between EPC levels and mortality in PAD patients. Methods EPC were measured in 367 PAD patients (age 69.22±10.3, 66.5% male, Fontaine stage I-II) by flow cytometry using the cell surface marker CD34+ and CD309+. Patients were followed for seven years to assess all cause and cardiovascular mortality. Patients were categorized into quartiles according to EPC levels for further analyses. Statistics included Kaplan-Meier and Cox regression. Results 89 patients died over the observation period. ICD-codes indicated a cardiovascular cause in 58 patients. The group with the highest count of EPC showed a trend towards higher all-cause mortality (p=0.070) and a significant association with cardiovascular mortality (p=0.002). Multivariable adjustment for age, c-reactive protein, systolic blood pressure, renal function (creatinine and urinary albumin), low density lipoprotein cholesterol, HbA1c, and smoking status revealed the EPC quartile to be an independent risk factor for cardiovascular mortality (p=0.016). Conclusion Increased levels of CD34+CD309+ cells are independently associated with long-term cardiovascular mortality in PAD patients.

AIMS Glycosylated acetyls (GlycA), a systemic marker of inflammation, were associated both with incident type 2 diabetes mellitus (T2DM) and incident cardiovascular (CV) disease. This study evaluates the predictive value of GlycA for long-term survival in patients with T2DM and peripheral artery disease (PAD). METHODS GlycA (mmol/l) levels were measured by nuclear magnetic resonance spectroscopy in a cross-sectional cohort of patients with PAD (n = 319). Both all-cause and CV mortality were evaluated after a follow-up of 9.0 (IQR 6.5-9.5) years. During the follow-up 117 patients died, of those 64 events were of CV origin (PAD-T2DM subgroup: all-cause mortality n = 60, CV-mortality n = 32). RESULTS PAD-T2DM showed a tendency towards a worse CV risk factor profile and a higher percentage of known coronary artery disease (24.9% vs 43.5%, p

Introduction: Angiopoietin 2 (ANG2) is a pro-inflammatory protein which is elevated in several inflammatory conditions and associated with increased morbidity and mortality. Hypothesis: ANG2 is associated with the composite of death, non-fatal myocardial infarction and non-fatal stroke (MACE) in patients with peripheral arterial disease (PAD). Methods: We measured serum ANG2 levels at baseline in 363 consecutive PAD patients with using multiplex bead array technology. Follow-up was five years. In Cox regression, confounding was evaluated by examining the relative change in coefficients (delta-beta) for ANG2 after the omission of the respective potential confounder from the fully adjusted model. Significant Confounding was defined as a delta-beta > 10%. Results: Using receiver operating characteristics, ANG2 exhibited an area under the curve of 0.6 (95%CI: 0.522 - 0.678) for the proper discrimination between MACE and non-events. A cut-off of 3.74 ng/ml resulted in the highest possible combination of specifity (57.5%) and sensitivity (63.3%) leading to an unadjusted hazard ratio (HR) of 2.17 (95%CI: 1.28 - 3.67) in Cox regression (p=0.004). Adjusting for age, sex, diabetes, systolic blood pressure, smoking status and renal function lead to a HR of 1.89 (95%CI: 1.11 - 3.24) for ANG2 >3.74 ng/ml (p=0.020). The additional adjustment for c-reactive protein (CRP) resulted in a no more significant HR of 1.68 (95%CI: 0.97 - 2.92)for high ANG2 (p=0.067). Diabetes and CRP, both significant predictors of MACE in our cohort, proved to be significant confounders with concomitant delta-betas of 19.7% and 22.9%, respectively. Conclusions: We are the first to demonstrate that elevated ANG2 levels are interrelated with MACE in patients with PAD. Inflammation and glucose hemostasis seem to influence this association.

Introduction: Proteins which are involved in the bone metabolism have recently been discovered to have significant implications in the development of vascular disease and complications. Osteopontin (OPN) has been suggested as a biomarker of atherosclerosis and vascular calcification. Hypothesis: OPN levels may be used for risk stratification in patients with peripheral artery disease (PAD). Methods: 361 patients suffering from PAD were studied and followed-up for five years. 60 outcome events, defined as death, non-fatal myocardial infarction or non-fatal stroke, were observed. Multiplex bead array technology was used to measure serum levels of OPN at baseline. For basic analysis, the patients were divided into quartiles according to OPN levels. A Cox regression model was used to identify confounding variables. Results: A significantly different event-free survival could be observed between all groups (log-rank: p=0.049). When comparing the highest quartile to the rest of the cohort, a further increase in significance could be observed (log-rank: p=0.017). A cut-off point accommodating the most favorable sensitivity (50.0%) and specificity (66.4%) was determined for regression analysis. The hazard ratio was 2.13 (1.26-3.60) in the unadjusted model and reached 2.16 (1.21-3.84) after adjustment for age, gender, diabetes, low-density lipoprotein cholesterol, blood pressure, smoking, c-reactive protein and renal function. Conclusions: Our results suggest an association between OPN levels and the rate of cardiovascular events in high risk patients. In our observations this relation was independent of established risk factors.

Introduction: Trefoil factor 3 (TFF3), first described in the intestine has general growth but also angiogenic properties. Recently a role in cancer and inflammation was found. Classic cardiovascular risk factors have less predictive power for morbidity and mortality in patients with peripheral arterial disease (PAD) compared to patients with coronary heart disease (CHD). Hypothesis: Serum TFF3 levels are able to predict major adverse cardiovascular events (MACE) in patients with PAD. Methods: We applied bead array technology to assess the serum levels of TFF3 in 359 patients of a prospective, on-going, long-term follow-up PAD study. First MACE was used as outcome parameter, defined as occurrence of any death, non-fatal myocardial infarction or non-fatal stroke. Statistical analysis were performed including univariate correlations, Receiver Operating Characteristic (ROC) curves, students t-test , ANOVA, Kaplan-Meier (KM)-analysis and Cox-regression. Results: Patients with systemic atherosclerosis defined as concomitant PAD, CHD and cerebrovascular disease had significant higher levels of TFF3 (59.38±20.86 ng/ml) at baseline compared to patients with only one (47.22±18.10 ng/ml; p=0.001) or two (49.12±19.43 ng/ml; p=0.006) manifestations of atherosclerosis. KM-analysis demonstrated a significant lower event-free survival for the fourth quartile of TFF3 (71.1%) compared to the other (88.1%) (Log-rank: p Conclusions: Our data imply a significant association of serum Trefoil Factor 3 and the occurrence of MACE in patients with atherosclerosis. This association of Trefoil Factor 3 and MACE was unchanged by C-reactive protein. Whether the association of Trefoil Factor 3 is causal, or a consequence of advanced atherosclerosis should be investigated in future trials containing time courses of Trefoil Factor 3 and MACE.